Multiple hydrogen-bonding induced nonconventional red fluorescence emission in hydrogels

The development of unconventional long-wavelength fluorescent polymer hydrogels without using polycyclic aromatic hydrocarbons or extended π-conjugation is a fundamental challenge in luminescent materials owing to a lack of understanding regarding the spatial interactions induced inherent clustering-triggered emission under water-rich conditions. Inspired by the color change of protein astaxanthin as a result of heat-induced denaturation, we propose a thermodynamically driven strategy to develop red fluorescence (~610 nm) by boiling multiple hydrogen-bonded poly(N-acryloylsemicarbazide) hydrogels in a water bath. We reveal that thermodynamically driven conformational changes of polymer chains from isolated hydrogen bonding donor-acceptor structures to through-space interaction structures induce intrinsic fluorescence shifts from blue to red during clustering-triggered emission. The proposed multiple hydrogen-bonding supramolecular hydrogel shows good fluorescence stability, mechanical robustness, and 3D printability for customizable shaping. We provide a viable method to prepare nonconventional long-wavelength fluorescent hydrogels towards soft fluorescent devices without initially introducing any fluorescent components.

In this paper, Wang et al. propose a thermodynamics-driven strategy to achieve the delocalized Hbonding induced nonconvenfional red fluorescent emission in hydrogel.They discussed the fundamental mechanism of this emergent phenomenon in fluorescent hydrogel.Importantly, these contribufions show significant implicafions for deeply understanding the inherent clustering-triggered emission (CTE) when forming the spafial conjugafion in water surroundings.Moreover, the authors also verified the feasibility of 3D-printed red fluorescent hydrogels in applicafions such as fluorescence detecfion and soft robofics by grayscale digital light processing (g-DLP) technology.This work bridges an obvious knowledge gap and provide a new insight in understanding the inherent clustering-triggered emission (CTE) when forms the spafial conjugafion in water surrounding.Therefore, it can be considered for publicafion in Nature Communicafions, but some minor comments are sfill required before publicafion.1.In Fig. 2d, the PNASC100 °C hydrogel also exhibits a blue light emission region at approximately 425 nm, please provide addifional explanafion.2. The fluorescent color dependents on the temperature of post-heafing.It can be found that the PNASC65℃ hydrogel retained blue fluorescent properfies, while the PNASC85℃ hydrogel exhibited a sudden transifion to red fluorescence.It's a large wavelength shift from blue to red.The authors should further discuss this process.And meanwhile, we are curious as to why hydrogels only show the isolated wavelength shift (i.e., blue or red) instead of a transifion state for other middle wavelength.3.For the solvent stability of hydrogels, the paper only verifies the fluorescence stability of polymer powders.However, the fluorescence stability of hydrogel was not verified in detail.The supplementafion is recommended.4. In Supplementary Fig. 5, the elongafion at break of the PNASC100℃ hydrogel decreases after heafing.What's the reason for this? 5.For the Cell viability assay, the arficle does not menfion the type of cells used, and addifional clarificafion is needed to declare.

Reviewer #3 (Remarks to the Author):
I co-reviewed this manuscript with one of the reviewers who provided the listed reports.This is part of the Nature Communicafions inifiafive to facilitate training in peer review and to provide appropriate recognifion for Early Career Researchers who co-review manuscripts.

Reviewer #4 (Remarks to the Author):
Overall the arficle is poorly wriften and requires an extensive spelling and grammar check.p. 3: No reference is made of a crosslinker.The authors refer to the use of N-acryloylsemicarbazide in the presence of an inifiator to create a hydrogel.However, starfing from this monomer, no hydrogel networks can be formed.Instead only linear polymer chains can be formed with lack of stability in the presence of a solvent.Furthermore, the authors should elaborate on possible side reacfions occurring in DMSO as solvent when performing a radical-mediated polymerizafion.
Furthermore, the authors should evidence hydrogel stability by referring to gel fracfions obtained following polymerizafion.I would also recommend to analyze the amount of unreacted double bonds with HR-MAS NMR spectroscopy to complement gel fracfion assays.Materials & Methods p.17: What yield was obtained for the monomer synthesis?p.20: How was DLP opfimized?What was the resolufion/CAD-CAM mimicry observed?It is surprising that the authors did not require photo-absorber for DLP (since this is typically applied in DLP to ensure appropriate resolufion).The materials & methods secfion reports on cell viability assays, but no results are menfioned in the arficle body regarding the execufion of such experiments.
1.I have reviewed the authors' responses to reviewer 4, and it appears that they have adequately addressed the quesfions raised by this reviewer.
2. I appreciate the authors' efforts in addressing most of the concerns I raised (reviewer 1).However, I do not completely agree with the explanafion the authors provided for the luminescence mechanism (for the second issue).I kindly suggest the authors meficulously revise the descripfion of the luminescent mechanism in this work before publicafion.
While acknowledging the significance of hydrogen bonding in achieving excellent CTE, I strongly suggest that the authors avoid solely aftribufing their luminescence mechanism to the electron delocalizafion of interchain strong mulfiple H-bonding interacfions.If the delocalizafion of electrons through hydrogen bonding is indeed efficient, it raises the quesfion of why the luminescence quantum efficiency is so low in this hydrogen-bond-rich system and, meanwhile, why the ice is not emissive (who shows one of the strongest H-bond).It is evident that a substanfial number of heteroatoms parficipate in hydrogen bond formafion in this system, creafing a conducive environment for clusteroluminescence.However, it is worth nofing that a small fracfion of heteroatoms that have not formed hydrogen bonds may potenfially engage in n-n/π TSI.The contribufion of this aspect should not be overlooked, parficularly in a weak clusteroluminescence system.
In relafion to the experiments described by the authors (i.e., adding DMSO), it is important to acknowledge that the breaking of hydrogen bonds will simultaneously disrupt other weak interacfions (such as n-n TSI) due to the more flexible environment.Meanwhile, EtOH has a stronger ability to destroy H-bond than DMSO, but red luminescence is sfill observed in EtOH.Consequently, these experiments may not offer convincing evidence to support the dominant role of the H-bond in electron delocalizafion.
As a result, in the main text, it is preferable for the authors to aftribute the nonconvenfional red fluorescent emission to contribufions of various heteroatom-involved interacfions (including heteroatom-involved hydrogen bonds and n-n/π TSI), rather than qualitafively aftribufing it to a specific singular H-bonding contribufion.

Reviewer #2 (Remarks to the Author):
I have read this revised mascript very carefully, feeling the manuscript of the current verson is well prepared for publicafion.
I co-reviewed this manuscript with one of the reviewers who provided the listed reports.This is part of the Nature Communicafions inifiafive to facilitate training in peer review and to provide appropriate recognifion for Early Career Researchers who co-review manuscripts.
Reviewer #1 (Remarks to the Author): 1.I have reviewed the authors' responses to reviewer 4, and it appears that they have adequately addressed the questions raised by this reviewer.
2. I appreciate the authors' efforts in addressing most of the concerns I raised (reviewer 1).However, I do not completely agree with the explanation the authors provided for the luminescence mechanism (for the second issue).I kindly suggest the authors meticulously revise the description of the luminescent mechanism in this work before publication.
While acknowledging the significance of hydrogen bonding in achieving excellent CTE, I strongly suggest that the authors avoid solely attributing their luminescence mechanism to the electron delocalization of interchain strong multiple H-bonding interactions.If the delocalization of electrons through hydrogen bonding is indeed efficient, it raises the question of why the luminescence quantum efficiency is so low in this hydrogen-bond-rich system and, meanwhile, why the ice is not emissive (who shows one of the strongest H-bond).It is evident that a substantial number of heteroatoms participate in hydrogen bond formation in this system, creating a conducive environment for clusteroluminescence.However, it is worth noting that a small fraction of heteroatoms that have not formed hydrogen bonds may potentially engage in n-n/π TSI.The contribution of this aspect should not be overlooked, particularly in a weak clusteroluminescence system.
In relation to the experiments described by the authors (i.e., adding DMSO), it is important to acknowledge that the breaking of hydrogen bonds will simultaneously disrupt other weak interactions (such as n-n TSI) due to the more flexible environment.
Meanwhile, EtOH has a stronger ability to destroy H-bond than DMSO, but red luminescence is still observed in EtOH.Consequently, these experiments may not offer convincing evidence to support the dominant role of the H-bond in electron delocalization.
As a result, in the main text, it is preferable for the authors to attribute the nonconventional red fluorescent emission to contributions of various heteroatominvolved interactions (including heteroatom-involved hydrogen bonds and n-n/π TSI), rather than qualitatively attributing it to a specific singular H-bonding contribution.natural light and UV light (365 nm).The as-PNASCDMSO-0% was heated at 100℃ for 48 h and named PNASCDMSO-0%.The scale bar is 5 mm.Supplementary Fig. 19 | Interaction of cluster luminescence in PNASCred hydrogels.
We added this result in Supplementary Fig. 19 and the revised description of the luminescent mechanism has been added in the revised manuscript.The revision made was as follows: In the "Main" section "Besides, a small fraction of heteroatoms (O and N) containing lone-pair electrons that do not form strong H-bonds may also participate in n-n, n-π interactions of TSI." In the "Luminescence mechanism of the hydrogels" section "In addition, it is also worth noting that a substantial number of heteroatoms participate in hydrogen bond formation in the hydrogel, which also plays a critical role in building a favorable environment for cluster luminescence.Besides, a small fraction of heteroatoms containing lone-pair electrons that do not form strong H-bonds may also participate in n-n, n-π interactions of TSI.So, the contribution of this part should not be overlooked (Supplementary Fig. 19)" In the "Conclusion" section "The electron-rich groups in the proposed hydrogel extended the electron delocalization through intermolecular charge transfer to achieve stable red fluorescence emission in a water environment by various heteroatom-involved interactions (including heteroatominvolved hydrogen bonds and n-n/π TSI)."

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): I am safisfied with the revised version and gladly recommend it to be published in Nature Communicafions.However, I sfill have a suggesfion about the fitle.I suppose that it is insufficient to only menfion the importance of hydrogen bonds in the fitle.There is no direct evidence that the delocalizafion of H-bonding contributes much to this nonconvenfional luminescence.To avoid academic controversy, I kindly suggested that it is befter to change the fitle "Delocalized H-bonding induced nonconvenfional red fluorescence emission in hydrogels" to " Mulfiple hydrogen-bonding induced nonconvenfional red fluorescence emission in hydrogels".

Reviewer #3 (Remarks to the Author):
I co-reviewed this manuscript with one of the reviewers who provided the listed reports.This is part of the Nature Communicafions inifiafive to facilitate training in peer review and to provide appropriate recognifion for Early Career Researchers who co-review manuscripts.